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Am J Physiol Gastrointest Liver Physiol 292: G201-G207, 2007. First published August 31, 2006; doi:10.1152/ajpgi.00186.2006
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LIVER AND BILIARY TRACT

Hepatocyte NF-{kappa}B activation is hepatoprotective during ischemia-reperfusion injury and is augmented by ischemic hypothermia

Satoshi Kuboki,1 Tomohisa Okaya,1 Rebecca Schuster,1 John Blanchard,1 Alvin Denenberg,2 Hector R. Wong,2 and Alex B. Lentsch1

1The Laboratory of Trauma, Sepsis and Inflammation Research, Department of Surgery, University of Cincinnati College of Medicine; and 2Division of Critical Care Medicine, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The present study examined the role of hepatocyte NF-{kappa}B activation during ischemia-reperfusion injury. Second, we evaluated the effects of ischemic hypothermia on NF-{kappa}B activation and liver injury. C57BL/6 mice underwent 90 min of partial hepatic ischemia and up to 8 h of reperfusion. Body temperature was regulated during the ischemic period between 35 and 37°C, 33 and 35°C, 29 and 33°C or unregulated, where temperature fell to <29°C. Liver injury, as measured by serum alanine aminotransferase as well as liver histopathology, was inversely proportional to regulated body temperature, with the unregulated group (<29°C) being highly protected and the normothermic group (35–37°C) displaying the greatest injury. Inflammation, as measured by production of TNF-{alpha} and liver recruitment of neutrophils, was greatest in the normothermic groups and lowest in the ischemic hypothermia groups. Interestingly, hepatocyte NF-{kappa}B activation was highest in the hypothermic group and least in the normothermic group. Paradoxically, degradation of I{kappa}B proteins, I{kappa}B-{alpha} and I{kappa}B-beta, was greatest in the normothermic group, suggesting an alternate NF-{kappa}B regulatory mechanism during ischemia-reperfusion injury. Subsequently, we found that NF-{kappa}B p65 protein was increasingly degraded in normothermic versus hypothermic groups, and this degradation was specific for hepatocytes and was associated with decreased expression of the peptidyl-prolyl isomerase Pin1. The data suggest that NF-{kappa}B activation in hepatocytes is a protective response during ischemia-reperfusion and can be augmented by ischemic hypothermia. Furthermore, it appears that Pin1 promotes NF-{kappa}B p65 protein stability such that decreased expression of Pin1 during ischemia-reperfusion results in p65 degradation, reduced nuclear translocation of NF-{kappa}B, and enhanced hepatocellular injury.

liver inflammation; nuclear factor-{kappa}B p65; Pin1

ISCHEMIA-REPERFUSION (I/R) injury of the liver is a primary complication of liver resection surgery, transplantation, and trauma. Hepatic I/R leads to an acute inflammatory response that may result in severe hepatocellular damage and organ dysfunction (10, 20, 22). This inflammatory response is characterized by early production of proinflammatory cytokines, such as TNF-{alpha}, which propagate the inflammatory response by upregulating vascular endothelial cell adhesion molecules and increasing the production of neutrophil chemoattractants, including CXC chemokines (4–6). The cooperative effects of adhesion molecules and CXC chemokines results in the recruitment of neutrophils from the vascular space into the hepatic parenchyma (12). These activated neutrophils then damage liver parenchymal cells through their release of reactive oxygen species and proteases (12, 13). In experimental models, blockade of inflammatory mediators has proven effective against liver injury after hepatic I/R (4–6, 8, 17), suggesting that interventional therapies designed to suppress the inflammatory response may have significant therapeutic benefits.

We (16) previously demonstrated that whole body hypothermia is protective against liver I/R injury in a manner associated with a diminished inflammatory response. Our study (16) demonstrated that low body temperature during the periods of ischemia and early reperfusion reduced the gene transcription and ultimate expression of a number of proinflammatory mediators. A potential contributing factor to the observed decrease in cytokine expression was reduced activation of JNK and activator protein (AP)-1 (16).

Another transcription factor that is tightly linked with the regulation of proinflammatory mediators during liver I/R injury is NF-{kappa}B (17, 21, 31). Currently, the cell-specific functions of NF-{kappa}B in the liver during I/R injury are not well understood. It is thought that NF-{kappa}B activation in Kupffer cells, hepatocytes, and sinusoidal endothelial cells results in the transcriptional activation of many proinflammatory genes (9, 19, 25–27). Alternatively, NF-{kappa}B activation in hepatocytes has been shown to be protective against some forms of injury (1–3, 11, 30). However, the precise function of this transcription factor in hepatocytes during I/R injury has not been determined. In the present study, we examined the activation of NF-{kappa}B in hepatocytes during I/R injury and explored the manner in which different degrees of ischemic hypothermia regulated this activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hepatic I/R injury model. Male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) weighing 23–27 g were used in all experiments. This project was approved by the University of Cincinnati Animal Care and Use Committee and was in compliance with National Institutes of Health guidelines. The animals underwent either sham surgery or I/R. In the latter group, body temperature was maintained in four different ranges during hepatic ischemia: 1) 35–37°C, 2) 33–35°C, 3) 29–33°C, and 4) unregulated temperature, where rectal temperatures fell to <29°C. Partial hepatic ischemia was induced as previously described (17). Briefly, mice were anesthetized with pentobarbital sodium (60 mg/kg ip). A midline laparotomy was performed, and an atraumatic clip was used to interrupt blood supply to the left lateral and median lobes of the liver. The caudal lobes retained intact portal and arterial inflow and venous outflow, preventing intestinal venous congestion. After 90 min of partial hepatic ischemia, the clip was removed, initiating hepatic reperfusion. The animals received 0.2 ml of sterile saline subcutaneously, the wound was closed in layers with 4-0 silk, and the animal was allowed to recover. Sham control mice underwent the same protocol without vascular occlusion. Rectal temperature was measured using an electronic thermometer with a probe (Fisher Scientific, Pittsburgh, PA) every 15 min after the injection of anesthesia. Body temperature was regulated with a heating pad under the recovery cage.

EMSA. Nuclear extracts of liver tissue were prepared by the method of Deryckere and Gannon (7) and analyzed by EMSA. Briefly, double-stranded NF-{kappa}B consensus oligonucleotide (Promega, Madison, WI) was end labeled with [{gamma}-32P]ATP (3,000 Ci/mmol at 10 mCi/ml; Amersham. Arlington Heights, IL). Binding reactions contained equal amounts of nuclear protein extract (20 µg) and 35 fmol (~50,000 counts/min, Cherenkov counting) of oligonucleotide and were incubated at room temperature for 30 min. Binding reaction products were separated in a 4% polyacrylamide gel and analyzed by autoradiography.

Western blot analyses. Liver samples were homogenized in lysis buffer [10 mM HEPES (pH 7.9), 150 mM NaCl, 1 mM EDTA, 0.6% Nonidet P-40, 0.5 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotonin, 10 µg/ml soybean trypsin inhibitor, and 1 µg/ml pepstatin] on ice. Homogenates were sonicated and centrifuged at 5,000 rpm to remove cellular debris. Hepatocytes and Kupffer cells were isolated as previously described (19). Protein concentrations of whole liver lysates or cell lysates were determined as described for nuclear extracts. Samples were separated in a denaturing 10% polyacrylimide gel and transferred to a 0.1-µm-pore nitrocellulose membrane. Nonspecific binding sites were blocked with Tris-buffered saline [TBS; 40 mM Tris (pH 7.6) and 300 mM NaCl] containing 5% nonfat dry milk for 12 h at 4°C. Membranes were then incubated with antibodies to I{kappa}B-{alpha}, I{kappa}B-beta, phospho-I{kappa}B-{alpha}, p65, or Pin1 (Santa Cruz Biotechnology, Santa Cruz, CA) in TBS with 0.1% Tween 20. Membranes were washed and incubated with secondary antibodies conjugated to horseradish peroxidase. Immunoreactive proteins were detected by enhanced chemiluminescence.

Liver neutrophil accumulation. Liver MPO content was assessed by methods described elsewhere (24). Briefly, liver tissue (100 mg) was homogenized in 2 ml of buffer A (3.4 mmol/l KH2HPO4 and 16 mmol/l Na2HPO4; pH 7.4). After being centrifuged for 20 min at 10,000 g, the pellet was resuspended in 10 vol of buffer B (43.2 mmol/l KH2HPO4, 6.5 mmol/l Na2HPO4, 10 mmol/l EDTA, and 0.5% hexadecyltrimethylammonium, pH 6.0) and sonicated for 10 s. After being heated for 2 h at 60°C, the supernatant was reacted with 3,3',3,5'-tetramethylbenzidine, and the optical density was read at 655 nm.

Blood and tissue analysis. Blood was obtained by cardiac puncture for analysis of serum alanine aminotransferase (ALT) as an index of hepatocellular injury. Measurements of serum ALT were made using a diagnostic kit (Sigma Chemical, St. Louis, MO). Serum levels of TNF-{alpha} were measured by sandwich ELISA according to the manufacturer's instructions (R&D Systems, Minneapolis, MN). Ischemic lobes (or corresponding lobes in the sham group) were excised for tissue analysis. Tissue were fixed in 10% formalin and then embedded in paraffin for light microscopy. Sections were stained with hematoxylin and eosin for histological examination.

Immunocytochemical labeling. Liver tissues were fixed in 10% neutral buffered formalin and embedded in paraffin before being sectioned. Tissue sections were then deparaffinized, washed twice in PBS (pH 7.4), and blocked with Image-iT FX signal enhancer (Molecular Probes, Eugene, OR) for 30 min. After being washed in PBS, tissue sections were incubated with rabbit polyclonal anti-p65 antibody (Santa Cruz Biotechnology) overnight at 4°C. Sections were washed in PBS and then incubated with goat anti-rabbit IgG conjugated with Alexa Fluor 594 (Molecular Probes) for 2 h at room temperature protected from light. After a final PBS wash, sections were examined, and images were acquired on a Nikon PCM 2000 laser confocal scanning microscope.

Hepatocyte and Kupffer cell isolation. Hepatocytes were isolated by nonrecirculating collagenase perfusion through the portal vein. Livers were perfused in situ for 5 min with Ca2+- and Mg2+-free HBSS (pH 7.4) with 50 U/ml collagenase at a flow rate initiated at 8 ml/min and then immediately increased to 55 ml/min. The liver was excised, minced, and strained through a steel mesh. The dispersed hepatocytes were collected by centrifugation at 50 g for 2 min at 4°C. Cells were washed three times in Williams media, and viability was checked by trypan blue exclusion. Kupffer cells were isolated by nonrecirculating collagenase-protease perfusion through the portal vein. Livers were perfused in situ for 5 min with sterile Ca2+- and Mg2+-free Gey's solution (pH 7.4) with 100 U/ml collagenase and 10 U/ml protease I. The liver was excised, minced, and incubated with stirring in the collagenase-protease-Gey's solution at 37°C for 30 min. The dispersed cells were filtered through nylon mesh filters (70 µm) and collected by centrifugation at 50 g for 2 min at 4°C. Cells were washed two times in Gey's solution. Kupffer cells were contained in the supernatants from the above washes. Cells were pelleted by centrifugation at 500 g for 9 min, resuspended in sterile Ca2+- and Mg2+-free HBSS (pH 7.4), and subjected to fractionation by elutriation. Centrifugal elutriation was performed using a Beckman Coulter J20-XPI centrifuge with a JE 5.0 elutriator rotor at a constant speed of 3,200 rpm with stepwise increases in perfusion rates. Kupffer cells were collected at the 44 ml/min fraction. The resulting cell isolates were washed, and viability was checked by trypan blue exclusion.

Statistical analysis. All data are expressed as means ± SE. Data were analyzed with one-way ANOVA with a subsequent Student-Newman-Keuls test. Differences were considered significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of ischemic hypothermia on reperfusion injury. To determine whether different degrees of hypothermia altered the response to hepatic I/R, we established four temperature groups. For each group, rectal temperature was monitored every 15 min during hepatic I/R. Just before the induction of anesthesia, the average rectal temperature of all mice was 36.5 ± 0.09°C (Fig. 1). Body temperature was regulated during the ischemic period at 35–37°C, 33–35°C, or 29–33°C or unregulated (<29°C). During the ischemic period, the average rectal temperature for mice maintained in the 35–37°C group was 35.95 ± 0.46°C; for mice in the 33–35°C group, it was 33.78 ± 0.52°C; for mice in the 29–33°C group, it was 29.72 ± 0.28°C; and for mice in the <29°C group, it was 25.14 ± 1.89°C. Body temperatures were significantly different from one another during the ischemic period (P < 0.05). Upon reperfusion, body temperature in all mice was maintained between 35 and 37°C. Thus, the hypothermic period occurred only during the period of ischemia. Within 30 min of reperfusion, all groups had similar body temperatures.


Figure 1
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  		Fig. 1. Body temperature during hepatic ischemia and reperfusion. Body temperature was regulated during the period of ischemia in the following manner: unregulated (<29°C), 29–33°C, 33–35°C, or 35–37°C. In all groups, body temperature was normal within 1 h of reperfusion. Data are means ± SE with n = 6 per group.

 
To assess whether varying degrees of hypothermia had different effects on liver injury after I/R, we examined liver histopathology, liver content of MPO, and serum levels of ALT. As shown in Fig. 2A, liver histology was normal in normothermic sham-operated mice. However, after ischemia and 8 h of reperfusion, mice regulated at 35–37°C or 33–35°C during ischemia had widespread hepatocellular necrosis with massive infiltrates of neutrophils (Fig. 2, B and C, respectively). In contrast, mice whose body temperature was regulated between 29 and 33°C during the ischemic period had far less necrosis and little evidence of neutrophil accumulation (Fig. 2D). Mice with unregulated body temperature (<29°C) had liver histology that appeared normal (Fig. 2E). Consistent with these observations were the biochemical assays of neutrophil accumulation and liver injury. There was a temperature-dependent increase for both parameters. For liver MPO content, little change from sham controls was measured in both <29°C and 29–33°C groups (Fig. 3, top). However, in 33–35°C and 35–37°C groups, liver MPO content increased with time and was significantly different from the <29°C and 29–33°C groups after 8 h of reperfusion (Fig. 3, top). An even more temperature-specific effect was observed with serum ALT values. Increases in serum ALT in the <29°C group were very modest at all reperfusion times (Fig. 3, bottom). Larger increases were observed in the 29–33°C group and even greater increases were detected in the 33–35°C and 35–37°C groups (Fig. 3, bottom).


Figure 2
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  		Fig. 2. Liver histopathology after ischemia and 8 h of reperfusion in sham-operated control mice (A) and in mice in which body temperature was maintained during ischemia at 35–37°C (B), 33–35°C (C), 29–33°C (D), or <29°C (E).

 

Figure 3
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  		Fig. 3. Effect of ischemic hypothermia on liver neutrophil accumulation and injury. Liver neutrophil accumulation was assessed by liver myeloperoxidase (MPO) content and hepatocellular injury was determined by serum alanine aminotransferase (ALT). Data are means ± SE with n = 6 per group with *P < 0.05 compared with 33–35°C and 35–37°C groups.

 
Effects of ischemic hypothermia on liver NF-{kappa}B activation. Our previous study (16) of the effects of hypothermia on liver inflammatory injury after I/R found increased activation of JNK and AP-1 in hypothermic mice. Because NF-{kappa}B is known to be central to both the inflammatory response and hepatoprotection, we examined the activation of this transcription factor under different degrees of hypothermia during ischemia. Whole liver nuclear extracts were subjected to EMSA. After 1 h of reperfusion, NF-{kappa}B activation was increased in all groups, but activation in the 35–37°C group was significantly lower than the other groups (Fig. 4). After 4 or 8 h of reperfusion, liver NF-{kappa}B activation in <29°C and 29–33°C groups remained very high and was significantly greater than the 33–35°C and 35–37°C groups, which decreased toward sham levels (Fig. 4). The NF-{kappa}B complex was found to be comprised primarily of p50-p65 heterodimers with p65-p65 dimers also present. These results are consistent with our previous report (15) of NF-{kappa}B composition. In support of these findings, immunofluorescence staining of NF-{kappa}B p65 in liver sections showed mild staining in both the cytoplasm and nuclei of hepatocytes in sham-operated control mice (Fig. 5). In mice with body temperature <29°C during ischemia, there was strong, distinct nuclear translocation of NF-{kappa}B p65 in hepatocytes after 1 h of reperfusion (Fig. 5). In contrast, NF-{kappa}B p65 staining in livers from the 35–37°C group was completely cytoplasmic, with no nuclear staining observed (Fig. 5).


Figure 4
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  		Fig. 4. Effect of ischemic hypothermia on liver NF-{kappa}B activation after reperfusion. A: liver nuclear extracts from mice undergoing sham surgery or ischemia (<29°C, 29–33°C, 33–35°C or 35–37°C) and reperfusion were subjected to electrophoretic mobility shift assay. The NF-{kappa}B complexes (p50/p65 and p65/p65) were quantitated by image analysis of autoradiograms. Data are means ± SE with n = 4 per group with P < 0.05 compared with: *all other temperature groups, {dagger}35–37°C group, {ddagger}33–35°C and 35–37°C groups. B: liver nuclear extracts from mice undergoing ischemia (29–33°C) and 1 h of reperfusion was subjected to supershift assays. Arrowheads indicate supershifts of p50 and p65.

 

Figure 5
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  		Fig. 5. Subcellular localization of NF-{kappa}B p65 in liver sections after sham surgery, or ischemia (<29°C or 35–37°C) and 1 h of reperfusion. NF-{kappa}B p65 was visualized using immunofluorescence staining. Negative control represents staining of liver sections with secondary antibody only.

 
Expression of TNF-{alpha} is regulated primarily by NF-{kappa}B, and Kupffer cell-produced TNF-{alpha} is known to be the primary inflammatory cytokine in this model (6, 28). Therefore, we examined serum levels of TNF-{alpha} after 1 h of reperfusion, a time at which we and others have shown that serum TNF-{alpha} is maximal (6, 25). Consistent with the liver neutrophil and injury results, serum levels of TNF-{alpha} were nearly undetectable in the <29°C group, and with increasing body temperature toward normothermia, there was significantly increased expression of TNF-{alpha} (Fig. 6). These data are consistent with the hypothesis that NF-{kappa}B activation in Kupffer cells is lowest in the <29°C group and greatest in the 35–37°C group.


Figure 6
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  		Fig. 6. Effects of ischemic hypothermia on serum levels of TNF{alpha} after 1 h of reperfusion. Serum TNF{alpha} was measured by ELISA. Data are means ± SE with n = 5 per group with *P < 0.05 compared with<29°C group.

 
Paradoxical degradation of I{kappa}B proteins. Because NF-{kappa}B activation is regulated by I{kappa}B proteins, we assessed the degradation of I{kappa}B proteins, I{kappa}B-{alpha} and I{kappa}B-beta. For both I{kappa}B proteins, there was a temperature-dependent effect on degradation that was contrary to NF-{kappa}B activation. Livers from mice in the <29°C and 29–33°C groups, which had the highest activation of NF-{kappa}B (Fig. 4), showed little (29–33°C) or no (<29°C) degradation of I{kappa}B-{alpha} or I{kappa}B-beta after 1 h of reperfusion (Fig. 7). In contrast, the 33–35°C and 35–37°C groups, which had the lowest levels of NF-{kappa}B activation (Fig. 4), displayed marked degradation of I{kappa}B-{alpha} or I{kappa}B-beta after 1 h of reperfusion (Fig. 7). Similar results were found after 4 or 8 h of reperfusion (Fig. 7). In agreement with these data was our finding that I{kappa}B-{alpha} from mice in the <29°C group was not serine phosphorylated (Fig. 8). However, I{kappa}B-{alpha} from mice in the 35–37°C group was highly serine phosphorylated (Fig. 8).


Figure 7
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  		Fig. 7. Effects of ischemic hypothermia on degradation of I{kappa}B{alpha} and I{kappa}Bbeta in liver after reperfusion. Liver lysates were assessed for I{kappa}B protein expression by Western blot. Chemiluminescence films were quantitated by image analysis. Data are means ± SE with n = 4 per group with P < 0.05 compared with: *all other temperature groups, {dagger}35–37°C group, {ddagger}33–35°C and 35–37°C groups.

 

Figure 8
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  		Fig. 8. Effects of ischemic hypothermia on serine phosphorylation of I{kappa}B{alpha} after 1 h of reperfusion. Liver lysates were assessed for phosphorylation of serine 32/36 of I{kappa}B{alpha} by Western blot. Chemiluminescence films of phosphorylated I{kappa}B{alpha} were quantitated by image analysis. Data are means ± SE with n = 3–4 per group. *P < 0.05 compared with all other groups.

 
NF-{kappa}B p65 degradation and Pin1 expression. To further explore the mechanism by which ischemic hypothermia promoted NF-{kappa}B activation in hepatocytes in the absence of I{kappa}B protein degradation, we examined the expression of NF-{kappa}B p65 in the <29°C and 35–37°C groups. In liver lysates, we found that protein expression of p65 was diminished in livers from the 35–37°C group compared with livers from the <29°C group. An analysis of the ischemic period showed that after just 45 min of ischemia, there was a marked reduction in p65 protein expression in livers from the 35–37°C group (Fig. 9A, top). These effects were also observed after 90 min of ischemia (Fig. 9A, top). After ischemia and 1 h of reperfusion, we found that p65 protein expression was inversely related to body temperature during ischemia, with progressively less p65 detected as body temperature increased from <29°C to 35–37°C (Fig. 9A, bottom). The stability of p65 has been shown to be regulated by the peptidyl-prolyl isomerase Pin1 (23). To determine whether Pin1 was responsible for the changes in p65 expression, we assessed Pin1 expression. Western blot analysis demonstrated that the expression of Pin1 mirrored that of p65, during both the ischemic (Fig. 9A, top) and postischemic (Fig. 9A, bottom) periods. Immunoprecipitation of p65 from liver lysates and Western blot analysis for Pin1 demonstrated a direct physical interaction between p65 and Pin1 and that this interaction was greatly reduced after ischemia and 1 h of reperfusion under normothermic, but not hypothermic, conditions (Fig. 9B). The reduced expression of p65 and Pin1 observed in normothermic groups was not due to global protein degradation. Western blot analysis of the unrelated protein c-Jun demonstrated that the expression of this protein was actually increased in normothermic livers (Fig. 10). These data suggest that the maintained expression of Pin1 during hypothermic ischemia stabilizes p65, thereby facilitating increased NF-{kappa}B activation.


Figure 9
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  		Fig. 9. Effects of ischemic hypothermia on expression of NF-{kappa}B p65 and the peptidyl-prolyl isomerase, Pin1. A: Western blots for p65 and Pin1 were performed on liver lysates obtained from the ischemic period of mice in the <29°C and 35–37°C groups and from all temperature groups after ischemia and 1 h of reperfusion. Results were quantitated by image analysis of autoradiograms. Data are means ± SE with n = 3 per group with P < 0.05 compared with: *all other temperature groups, {dagger}35–37°C group, {ddagger}33–35°C and 35–37°C groups. B: immunoprecipitation of NF-{kappa}B p65 and subsequent Western blot of Pin1. Data are representative of three independent experiments.

 

Figure 10
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  		Fig. 10. Effects of hypothermia on expression of c-Jun. Liver lysates from mice after ischemia and 1 h of reperfusion were Western blotted for c-Jun and beta-actin as a loading control. Data are representative of three independent experiments.

 
Cell-specific expression of NF-{kappa}B p65 and Pin1. Because reduced NF-{kappa}B activation was observed in hepatocytes (Fig. 5), but production of TNF-{alpha} was elevated (Fig. 6) in normothermic livers, we assessed the cell-specific expression of p65 and Pin1 to determine whether NF-{kappa}B activation may be differentially activated in hepatocytes compared with Kupffer cells. In isolated hepatocytes, the expression of p65, Pin1, and I{kappa}B-{alpha} was identical to that found in whole liver extracts (Fig. 11). In Kupffer cells, the expression of p65 was not reduced in the 35–37°C group (Fig. 11). In fact, it appeared that there was slightly higher p65 expression in the 35–37°C group compared with the <29°C group. Kupffer cell expression of Pin1 was not different between the <29°C and 35–37°C groups (Fig. 11). Finally, the expression of I{kappa}B-{alpha} in Kupffer cells was decreased in the <29°C group but was much lower still in the 35–37°C group (Fig. 11). These findings indicate that NF-{kappa}B activation in Kupffer cells occurs under both normo- and hypothermic conditions but is far more robust in normothermic livers, consistent with greater production of TNF-{alpha} in these groups. These data strongly suggest that in Kupffer cells, Pin1 expression is maintained and p65 is not degraded, resulting in normal activation of NF-{kappa}B and the expression of proinflammatory mediators.


Figure 11
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  		Fig. 11. Hepatocyte and Kupffer cell expression of NF-{kappa}B p65, Pin1 and I{kappa}B{alpha} after ischemia and 1 h of reperfusion. Protein extracts from hepatocytes and Kupffer cells isolated from mice undergoing ischemia/reperfusion in the <29°C and 35–37°C groups were subjected to Western blot. Data are representative of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The role of NF-{kappa}B during hepatic I/R injury has long been thought to be purely proinflammatory in nature. Many studies, including some of our past reports, have shown that interventions that reduce inflammatory injury in this setting also reduce NF-{kappa}B activation. It has therefore been postulated that the inhibition of NF-{kappa}B suppresses the inflammatory response and therefore limits injury. However, data from other models of liver injury have suggested that hepatocyte activation of NF-{kappa}B is cytoprotective (1–3, 11, 30). Thus, the functions of NF-{kappa}B in different liver cell populations are likely to be quite different and potentially opposing in nature. Our present data support this notion and demonstrate that the hepatoprotective effect of hypothermia during the ischemic period is associated with increased NF-{kappa}B activation selectively in hepatocytes. We found that NF-{kappa}B activation in whole livers (which is largely representative of hepatocytes) as well as in hepatocytes (by immunofluorescence) was increased in ischemic hypothermia groups, whereas those groups with more normal core temperatures had less hepatocyte NF-{kappa}B activation. This, along with the findings that the production of TNF-{alpha} was greatly increased in normothermic groups (33–35°C and 35–37°C groups) but very little in hypothermic groups, strongly suggests that Kupffer cell NF-{kappa}B activation is greater in the normothermic groups. Furthermore, we found that p65 expresssion was not degraded, but elevated, in isolated Kupffer cells from normothermic mice, suggesting that NF-{kappa}B activation in these cells is not affected by body temperature. Our laboratory (16) previously found that the production of proinflammatory mediators such as TNF-{alpha}, IL-1beta, and macrophage inflammatory protein-2 were greatly reduced in hypothermic animals undergoing hepatic I/R. Those findings are consistent with our present data and suggest that Kupffer cell NF-{kappa}B is suppressed by ischemic hypothermia. This apparent cell-specific dichotomous function of NF-{kappa}B implies that global modulation of this transcription factor may often not produce the desired therapeutic effect and that cell-targeted approaches may be required.

Another novel observation of the present study was the finding that NF-{kappa}B activation in hepatocytes during reperfusion injury was somewhat dissociated from the degradation of I{kappa}B proteins. This was especially apparent in livers of animals undergoing hypothermic ischemia. In normothermic groups, we found that the I{kappa}B protein I{kappa}B-{alpha} was serine phosphorylated and that both I{kappa}B-{alpha} and I{kappa}B-beta underwent marked degradation. Despite this, hepatocyte NF-{kappa}B activation was marginally increased over sham controls. Recently, another regulatory mechanism helping to govern NF-{kappa}B activation has been discovered. This mechanism involves the peptidyl-prolyl isomerase Pin1 (14, 29). Pin1 has been shown by Ryo et al. (23) to specifically bind to the pThr254-Pro motif in p65, inhibit its binding to I{kappa}B-{alpha}, facilitate its nuclear translocation, and enhance protein stability. This study further demonstrated that Pin1-deficient mice are refractory to NF-{kappa}B activation by cytokine signals and that the function of Pin1 is dependent on binding to the pThr254-Pro motif in p65, as a p65-T254A mutant was extremely unstable and unable to transactivate NF-{kappa}B target genes (23). In the present study, we also showed a direct interaction between Pin1 and p65. Normothermic mice, which had reduced activation of NF-{kappa}B, were found to have increased degradation of p65 protein that was accompanied by decreased expression of Pin1. In contrast, in livers from mice undergoing hypothermic ischemia, Pin1 expression was normal and there was no degradation of p65. These data suggest that alterations in Pin1 expression induced by I/R greatly affect the activation of NF-{kappa}B in hepatocytes such that low Pin1 expression is accompanied by decreased p65 stability and nuclear translocation despite I{kappa}B degradation. Thus, strategies that preserve of Pin1 expression during ischemia and/or reperfusion may represent a novel therapeutic strategy.

In summary, our study provides strong evidence to suggest that NF-{kappa}B activation in hepatocytes is a protective response during I/R. Furthermore, this response is augmented by ischemic hypothermia. More interestingly, we found that Pin1 appears to promote NF-{kappa}B p65 protein stability and that decreased expression of Pin1 during I/R results in degradation of p65, reduced nuclear translocation of NF-{kappa}B, and enhanced hepatocellular injury. These data suggest that Pin1 may be a critical regulator of NF-{kappa}B in pathological conditions and may represent a potential therapeutic target.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants AG-025881, DK-56029, and HL-72552.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. B. Lentsch, Dept. of Surgery, Univ. of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0558 (e-mail: alex.lentsch{at}uc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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S. Kuboki, R. Schuster, J. Blanchard, T. A. Pritts, H. R. Wong, and A. B. Lentsch
Role of heat shock protein 70 in hepatic ischemia-reperfusion injury in mice
Am J Physiol Gastrointest Liver Physiol, April 1, 2007; 292(4): G1141 - G1149.
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